Tissue scaffold

ABSTRACT

A tissue scaffold includes a first film having a plurality of cell openings and a second film adjacent the first film and having a plurality of cell openings larger than the cell openings of the first film. The cell openings of the first film interconnect with the cell openings of the second film to define pathways extending through the first and second films.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Ireland Patent Application2004/0751, filed on Nov. 9, 2004, the entire content of which is herebyincorporated by reference in the present application.

TECHNICAL FIELD

This application relates generally to cell and tissue scaffolds.

BACKGROUND

The field of tissue engineering has resulted in the development ofbiocompatible scaffolds with significant potential for use in the repairand regeneration of tissue. For example, the use of porous mesh plugscomposed of hydroxy acid polymers such as polylactide for healing bonevoids was described by Brekke (see U.S. Pat. No. 4,186,448). Open celltantalum structures are described by Kaplan (see U.S. Pat. No.5,282,861). Biodegradable and bioresorbable templates are created usingleachables as described by Mikos (see U.S. Pat. Nos. 5,522,895 and5,514,378). Multi-phase bioerodible implants and methods have beendescribed by Athanasiou (see U.S. Pat. No. 5,607,474). Scaffolds canalso be produced using vacuum foaming techniques as described by Brekke(see U.S. Pat. Nos. 5,755,792 and 5,133,755). Molded porousbiodegradable polymer implants can also be created as described byWalter (see U.S. Pat. No. 5,716,413). A biodegradable foam useful forcell transplantation is described by Leong (see U.S. Pat. No.5,686,091). A polymeric foam with continuous open cell pores containingliving cells is described by Shalaby (see U.S. Pat. No. 5,677,355). Thepreparation of a three-dimensional fibrous scaffold for attaching cellsto produce vascularized tissue in vivo is described by Vacanti (see U.S.Pat. No. 5,770,193). Textile based porous scaffolds have also beendescribed (see U.S. Pat. Nos. 5,770,193 and 5,711,960). A hernia meshwith two or more functional components or layers with differentdegradation rates is described by Tormala (see U.S. Pat. No. 6,319,264).Microfabricated membranes and matrices with a three-dimensionaltopography are described by Morgan (see U.S. Pat. No. 6,479,072). Foambased scaffolds have also been described by Vyakarnam (see EP 1452191A2and EP 1064958B1). Two layered structures based on cultured cells aredescribed by Murphy (see EP 1131410B1).

SUMMARY

The present invention features tissue scaffolds and methods of makingand using these scaffolds for tissue engineering. For example, thescaffolds can be configured to facilitate tissue regeneration (e.g.,bone or muscle formation) or to replace tissues such as adipose tissue(as may be required in cosmetic or reconstructive surgeries), bloodvessels and valves (as may be required in connection with angioplasties,vessel inflammation, or valve deterioration), or skin (as may berequired where the skin is damaged by heat or mechanical force or bydisease (e.g., by diabetic ulcers)). We tend to use the terms “tissuescaffold(s)” and “scaffold(s)” interchangeably. As the scaffolds areintended for use with patients, the materials from which they are madeare substantially non-toxic. Accordingly, we may also refer to“biocompatible (tissue) scaffolds”.

The scaffolds can include two or more films having properties that canbe varied to alter the tissue scaffold's features (e.g., strength, voidor “open space” volume, porosity, and durability) and performance. Oneor more of the films can include a plurality of cell openings, each ofwhich defines a pore. Cell openings within the plurality can vary insize and/or shape and may be uniform or non-uniform within a given filmor scaffold. For example, the tissue scaffold can have, or can include,a first film including a plurality of cell openings and a second film,adjacent the first film, that includes a plurality of cell openings thatvary in size, shape, or pattern from those of the first film (e.g., thecell openings of the second film can be of the same shape or pattern butlarger than the cell openings of the first film). In embodiments wherethe size of the cell openings vary, at least one of the first and secondfilms can include progressively larger cell openings along a radialdirection to define a cell opening gradient. Alternatively, at least oneof the first and second films can include a plurality of cell openingssized and configured to define a cell opening gradient along the film.Thus, the size and/or pattern of the cell openings can be altered togenerate a radial or axial porosity gradient within a film. Where sizeremains constant, an increase in the density of the cells openings canbe used to increase porosity or to generate a porosity gradient. In anyembodiment where a film includes progressively larger cell openings todefine a cell opening gradient, that gradient can be defined radially oraxially.

Moreover, the cell openings of the first film can interconnect with thecell openings of the second film to define pathways extending from thefirst film to the second film. We may refer to these pathways asinterconnecting pores or regions of interconnectivity. The regions ofinterconnectivity can be generated or altered by orienting or alteringthe orientation of any one film to another (e.g., the orientation of afirst film with respect to a second). The regions of interconnectivitycan be substantially identical within a scaffold or may vary as the cellpattern(s) within the films vary. For example, in a first orientation ofthe first film with respect to the second film, the cell openings of thefirst film can be aligned with the cell opening of the second film todefine a first plurality of pathways; in a second orientation of thefirst film with respect to the second film, the cell openings of thefirst film can be substantially offset from the cell openings of thesecond film to define a second plurality of pathways. In addition, thedesign of the cell opening pattern of the first film or a first pair orgroup of films can be the same as or different from the design of thecell opening pattern of the second film or a second pair or group offilms. The porosity of one scaffold can be the same as that of another,even where the orientation of the films within the scaffolds isdifferent.

The tissue scaffolds can also include a plurality of delivery channels(which we may refer to more simply as “channels”) extending from thefirst film to the second film. Like the cell openings and the pores theydefine, the delivery channels can vary in size, shape, or pattern andcan be uniform or non-uniform within a given film or scaffold.

At least one of the first and second films, and up to all of the filmswithin a scaffold, can also include features to align the cell openingsof the first and second films when joined together. The films can bejoined thermally, mechanically (e.g., by a suture or staple) orchemically (e.g., by a biocompatible adhesive).

The films can assume essentially any shape. For example, the first andsecond films can be substantially identical in their dimensions (asmeasured, e.g., by length, width, or circumference) and can besubstantially circular, oval, square, rectangular, triangular,hexagonal, or irregular in outline.

The films can be manufactured from a variety of materials, which may ormay not be substantially identical and may or may not be bioabsorbable.For example, the tissue scaffolds can have, or can include, a first filmcomprised of a first material and a second film comprised of a secondmaterial. Where the materials are bioabsorbable, the first material canhave a higher absorption rate than the second material. The materialswithin a film (e.g., polymers and copolymers) can also be oriented withrespect to one another. For example, one can apply heat and a mechanicalload to orient the polymers within a film. The oriented film isstronger, and may be exponentially stronger, than a non-oriented film.

The tissue scaffolds can further include one or more therapeutic agents(e.g., growth factors), which may be included in at least one of thecell openings, pathways, or channels within a film or plurality offilms. Alternatively, or in addition, the scaffolds can further includeone or more types of biological cells (e.g., stem cells, progenitorcells (e.g., osteoblasts or any other partially differentiated cell),cells of an established cell line, or mature cells such as fibroblasts),which may be included in at least one of the cell openings, pathways, orchannels within a film or plurality of films. The tissue scaffolds canalso include one or more antibiotics, antiviral agents, or antifungalagents, or a combination thereof, and/or one or more vitamins orminerals. Biological cells can also be included. For example, the cellopenings of the first and second films can be sized and configured todefine multiple cell opening gradients to establish pathways forpreferential cell placement, culturing (or growth), or ingrowth (e.g.,from a tissue within a patient to whom the scaffold is adminstered).While therapeutic agents are described further below, we note here thatthey can include a naturally or nonnaturally-occurring material thatsubstantially modifies (by suppressing or promoting) tissue adhesion.

As noted, growth factors can be incorporated into the interconnectingpores or channels of a scaffold. Suitable growth factors includecytokines, interleukins, and other peptide growth factors such asepidermal growth factor (EGF), members of the fibroblast growth factor(FGF) family, platelet-derived growth factor (PDGF), nerve growth factor(NGF), glial growth factor (GGF), vascular endothelial growth factor(VEGF), or members of the Transforming Growth Factor (TGF) family (e.g.,TGF-α or TGF-β).

The interconnecting pores, the channels, or one or more surfaces of ascaffold (e.g., a surface that comes into contact with cells, tissues,or organs upon implantation in a subject) can also contain or be coatedwith one or more molecules involved in cell-cell adhesion or cell-matrixadhesion (i.e., an adhesion ligand). The adhesion ligand can be anadheren or cadherin and, more specifically, can be of the ICAM(intercellular adhesion molecule) family or the N-CAM (neural celladhesion molecule) family of proteins. We expect the incorporation ofadhesion ligands as pendant functionalities into our scaffolds tofacilitate integrin-dependent migration of cells, such as fibroblastsand endothelial cells, to and into the scaffolds. Growth factors locatedtherein (some of which are exemplified above) could then induce thedesired differentiation and necessary mitotic effect. The growthfactor(s) could also facilitate proliferation and differentiation ofstem cells or progenitor cells included in the scaffold. Depending onwhether the scaffold is biodegradable or not, it can serve as either aprovisional or permanent matrix for in vivo tissue regeneration. Thus,the scaffolds of the invention can include, as therapeutic agents,ligands for cell adhesion; a mechanism of relatively rapid and localizedmatrix dissolution (the fibrin scaffold paradigm), ideally synchronizedto cellular invasion; morphogenic signals to attract and retainendogenous or exogenous progenitor cells and induce theirdifferentiation to a tissue specific pathway. Where the scaffold isbiodegradable, surgical removal should not be required (and the risksassociated with such surgery are avoided).

Regardless of the precise content or configuration of the films withinthe scaffold, at least one of the first and second films can include oneor more attachment regions configured to receive surgical fasteningelements or delivery devices (e.g., pipettes, needles, syringes, and thelike, through which agents such as those described herein can bedelivered to the scaffold).

In one embodiment, the invention features a tissue scaffold that has, orthat includes: a first film having a first porosity; a second filmjoined to the first film and including a second porosity less than orgreater than the first porosity; and a plurality of cell openingsextending through the first and second films. The first porosity and thesecond porosity define a porosity gradient extending from the first tothe second film to selectively promote cellular regeneration along thegradient.

The invention also features methods of repairing or engineering tissueand thereby treating a subject or “patient”. Accordingly, the inventionencompasses the use of a tissue scaffold as described herein in therepair or engineering of tissue. While we expect the methods will becarried out with human patients, the invention is not so limited. Thescaffolds can be administered to tissues of non-human animals such asmammals (e.g., dogs and cats) and birds. More specifically, one canapply a scaffold as described herein to a target tissue (e.g., muscle,connective tissue such as bone, cartilage, ligaments, and tendons, ablood vessel (including an interior surface of an artery or vein), thegastrointestinal tract, a subcutaneous space, or to the skin). Thescaffold can be applied in the course of a sterile surgical procedure inthe same or similar manner as presently available implants areadministered to tissues. Where the tissue scaffold includes films havingdiffering properties, one can position the scaffold so that certainfilm(s) contact a first target tissue and certain film(s) contact asecond target tissue. One or more of the properties of the film(s)contacting the first target tissue may better promote remodeling orrepair of that tissue, while one or more of the properties of thefilm(s) contacting the second target tissue may better promoteremodeling or repair of that second tissue. One can, for example,position a plurality of delivery channels extending from a first film orplurality of films to a second film or plurality of films to apredetermined region of a tissue or tissues.

The methods can further include a step in which a subject is identifiedor diagnosed as having a disease or condition that would benefit fromapplication of a tissue scaffold.

The methods can further include a step in which one introduces an agent(e.g., a therapeutic agent) to the tissue through the delivery channels.Cells can be similarly introduced, with or without a therapeutic agent.Where an agent or cells is/are introduced, one can apply a pressuredifferential across first and second ends of the delivery channels togenerate fluid flow therethrough.

The invention also features methods of making a tissue scaffold asdescribed herein. The methods can be carried out by forming cellopenings in a first film to define a first porosity (porosity beinggoverned by a plurality of pores defined by the openings); forming cellopenings in a second film to define a second porosity greater than thefirst porosity; aligning the first film with respect to the second film;and attaching the first and second films such that the cell openings ofthe first film interconnect with the cell openings of the second film todefine pathways extending from the first film to the second film. Thematerials within the films (e.g., the polymers and copolymers describedherein) can be oriented if desired by application of a mechanical forceor load to the film. Of course, our characterization of size and otherproperties is relative. In any embodiment, the size of one cell openingin one film may be greater than, less than, or substantially the same asthe size of another cell opening in another film. The same is true ofother properties such as porosity; the comparators are relative.

The cell openings can be formed by any acceptable process. For example,the cell openings can be formed using laser ablation, die punching,extrusion, injection molding, electrospinning or dip coating techniques.

One of the hurdles in achieving successful cell transplantation andtissue engineering can be the lack of adequate vacsularization. Inprocedures where that is a concern, the scaffolds described hereinhaving channels sufficiently large to accommodate blood vessel ingrowthcan be used. In those applications, the scaffold can also include one ormore therapeutic agents that promote blood vessel growth (e.g., VEGF).

The scaffolds and implants presently in use may be deficient in one ormore ways and, while the present invention is not so limited, thescaffolds described herein may have one or more advantages over thosepreviously described. For example, the present scaffolds can beconstructed in a way that produces pores and channels with controlleddimensions and the properties of the films that constitute the scaffoldscan be varied to predictably alter the scaffolds' characteristics inways that favorably impact healing and cellular responses. The presentscaffolds can also be configured to improve the delivery of nutrients,fluids, cells (whether autologous or xenogeneic), therapeutic agents andthe like. The architecture of the present scaffolds may also be morereadily controlled (e.g., from batch to batch). The porosity of currentscaffolds may be approximated, and the mechanical stress/strain profilescan be too high or too low.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features and advantages ofwill be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a plan view of a film used to make tissue scaffolds with 500micron cell opening patterns.

FIG. 1B is a side view of the film of FIG. 1A used to make tissuescaffolds with 500 micron cell opening patterns.

FIG. 1C is a plan view of a film used to make tissue scaffolds with 750micron cell opening patterns.

FIG. 1D is a side view of the film of FIG. 1C used to make tissuescaffolds with 750 micron cell opening patterns.

FIG. 1E is a perspective view of two films used to make tissue scaffoldswith different offset cell opening patterns.

FIG. 1F is a plan view of two films used to make tissue scaffolds withdifferent cell opening patterns offset combined to create a 250 microninterconnecting pore.

FIG. 1G is a side view of the two films of FIG. 1F used to make tissuescaffolds with different cell opening patterns combined to create a 250micron interconnecting pore.

FIG. 1H is a perspective view of six films used to make tissue scaffoldswith different cell opening patterns.

FIG. 1I is a perspective view of six films used to make tissue scaffoldswith different cell opening patterns combined to create a 250 microninterconnecting pore.

FIG. 1J is a side view of the six films of FIG. 1I used to make tissuescaffolds with different cell opening patterns combined to create a 250micron interconnecting pore.

FIG. 1K is a perspective view of ten films used to make tissue scaffoldswith different cell opening patterns.

FIG. 1L is a perspective view of the ten films of FIG. 1K used to maketissue scaffolds with different cell opening patterns combined to createa 250 micron interconnecting pore.

FIG. 1M is a side view of six films used to make tissue scaffolds withdifferent cell opening patterns combined to create a 250 microninterconnecting pore.

FIG. 1N is a plan view of ten films used to make tissue scaffolds withdifferent cell opening patterns combined to create a 250 microninterconnecting pore.

FIG. 1O is a perspective view of ten films used to make tissue scaffoldswith different cell opening patterns combined to create a 250 microninterconnecting pore in partial section.

FIG. 2A is a plan view of a film used to make tissue scaffolds with 1000micron round cell opening patterns.

FIG. 2B is a side view of the film of FIG. 2A used to make tissuescaffolds with 1000 micron round cell opening patterns.

FIG. 2C is a plan view of a film used to make tissue scaffolds with 1000micron round cell opening patterns.

FIG. 2D is a side view of the film of FIG. 2C used to make tissuescaffolds with 1000 micron round cell opening patterns.

FIG. 2E is a perspective view of two films used to make tissue scaffoldswith offset cell opening patterns.

FIG. 2F is a perspective view of six films used to make tissue scaffoldswith offset cell opening patterns.

FIG. 2G is a perspective view of six films used to make tissue scaffoldswith 1000 micron cell opening patterns offset by 750 microns andcombined to create a 250 micron interconnecting pore.

FIG. 2H is a perspective view of the six films of FIG. 2F used to maketissue scaffolds with 1000 micron cell opening patterns offset by 750microns and combined to create a 250 micron interconnecting pores inpartial section.

FIG. 3A is a plan view of a film used to make tissue scaffolds with 300to 1300 micron round and oval cell opening patterns.

FIG. 3B is a side view of the film of FIG. 3A used to make tissuescaffolds with 300 to 1300 micron round and oval cell opening patterns.

FIG. 3C is a plan view of a film used to make tissue scaffolds with 300to 1300 micron round and oval cell opening patterns.

FIG. 3D is a side view of the film of FIG. 3C used to make tissuescaffolds with 300 to 1300 micron round and oval cell opening patterns.

FIG. 3E is a plan view of a film used to make tissue scaffolds with 300to 1300 micron round and oval cell opening patterns.

FIG. 3F is a side view of the film of FIG. 3E used to make tissuescaffolds with 300 to 1300 micron round and oval cell opening patterns.

FIG. 3G is a plan view of a film used to make tissue scaffolds with 300to 1300 micron round and oval cell opening patterns.

FIG. 3H is a side view of the film of FIG. 3G used to make tissuescaffolds with 300 to 1300 micron round and oval cell opening patterns.

FIG. 3I is a plan view of a film used to make tissue scaffolds with 300to 1300 micron round and oval cell opening patterns.

FIG. 3J is a side view of the film of FIG. 3I used to make tissuescaffolds with 300 to 1300 micron round and oval cell opening patterns.

FIG. 3K is a perspective view of five films used to make tissuescaffolds with offset cell opening patterns.

FIG. 3L is a perspective view of the five films of FIG. 3K combined tomake tissue scaffolds with offset cell opening patterns.

FIG. 3M is a perspective view of five films used to make tissuescaffolds with 300 to 1300 micron cell opening patterns offset andcombined to create an interconnecting pores in partial section.

FIG. 4A is a plan view of a film used to make tissue scaffolds with 60to 950 micron oval cell opening patterns with a gradient pattern.

FIG. 4B is a side view of the film of FIG. 4A used to make tissuescaffolds with 60 to 950 micron round and oval cell opening patternswith a gradient pattern.

FIG. 4C is a plan view of a film used to make tissue scaffolds with 270to 2080 micron round and oval cell opening patterns with a gradientpattern.

FIG. 4D is a side view of the film of FIG. 4C used to make tissuescaffolds with 270 to 2080 micron round and oval cell opening patternswith a gradient pattern.

FIG. 4E is a perspective view of two films used to make tissue scaffoldswith offset cell opening patterns with a gradient pattern.

FIG. 4F is a plan view of two films combined to make tissue scaffoldswith offset cell opening patterns with a gradient pattern to create aninterconnecting pore scaffold with a gradient in porosity.

FIG. 4G is a side view of the two films of FIG. 4F combined to maketissue scaffolds with offset cell opening patterns with a gradientpattern to create an interconnecting pore scaffold with a gradient inporosity.

FIG. 4H is a perspective view of ten films used to make tissue scaffoldswith offset cell opening patterns with a gradient pattern.

FIG. 4I is a perspective view of the ten films of FIG. 4H combined tomake tissue scaffolds with offset cell opening patterns with a gradientpattern to create an interconnecting pore scaffold with a gradient inporosity.

FIG. 5A is a perspective view of fifteen films used to make tissuescaffolds with offset cell opening patterns with a gradient pattern andtwo hydrogel films.

FIG. 5B is a plan view of the fifteen films of FIG. 5A combined to maketissue scaffolds with offset cell opening patterns with a gradientpattern to create an interconnecting pore scaffold with a gradient inporosity and two hydrogel films.

FIG. 5C is a perspective view of fifteen films combined to make tissuescaffolds with offset cell opening patterns with a gradient pattern tocreate an interconnecting pore scaffold with a gradient in porosity andtwo hydrogel films in partial section.

FIG. 6A is a perspective view of twenty seven films used to make atissue scaffold with delivery channel openings within the scaffold.

FIG. 6B is a plan view of the twenty five films of FIG. 6A combined tomake a tissue scaffold with delivery channel openings within thescaffold.

FIG. 6C is a side view of the twenty five films of FIG. 6A combined tomake a tissue scaffold with delivery channel openings within thescaffold.

FIG. 6D is a perspective view of the twenty five films of FIG. 6Acombined to make a tissue scaffold with delivery channel openings withinthe scaffold in partial section.

FIG. 6E is a perspective view of twenty seven films combined to make atissue scaffold with delivery channel openings within the scaffold witha cut out section and isolated on the top and bottom in partial section.

FIG. 6F is a perspective view of twenty seven films combined to make atissue scaffold with delivery channel openings within the scaffold inpartial section to depict the process of delivering agents or cells tothe channel openings.

FIG. 6G is a perspective view of twenty seven films combined to make atissue scaffold with delivery channel openings within the scaffold inpartial section to depict the process of delivering agents or cells tothe channel openings.

FIG. 6H is a perspective view of two separate tissue scaffolds withseparate delivery channel openings within each scaffold in partialsection to depict delivering agents or cells to the channel openings.

FIG. 6I is a perspective view of two separate tissue scaffolds combinedto make a composite tissue scaffold with separate delivery channelopenings within each scaffold in partial section depicting deliveringagents or cells to the channel openings.

FIG. 6J is a perspective view of twenty five films combined to make atissue scaffold with ingrowth channel openings within the scaffold thatextend to the center of the scaffold.

FIG. 6K is a perspective view of twenty five films combined to make atissue scaffold with ingrowth channel openings within the scaffold thatextend to the center of the scaffoldand depicting a blood vessel growinginto the channels of the scaffold.

FIG. 7A is a plan view of a film used to make tissue scaffolds with ovalcell opening patterns with a gradient pattern.

FIG. 7B is a side view of the film of FIG. 7A used to make tissuescaffolds with oval cell opening patterns with a gradient pattern.

FIG. 7C is a plan view of a film used to make tissue scaffolds with ovalcell opening patterns with a gradient pattern.

FIG. 7D is a side view of the film of FIG. 7C used to make tissuescaffolds with oval cell opening patterns with a gradient pattern.

FIG. 7E is a perspective view of two films used to make tissue scaffoldswith offset cell opening patterns with a gradient pattern.

FIG. 7F is a perspective view of six films used to make tissue scaffoldswith offset cell opening patterns with a gradient pattern.

FIG. 7G is a perspective view of the six films of FIG. 7F combined tomake tissue scaffolds with offset cell opening patterns with a gradientpattern to create an interconnecting pore scaffold with a gradient inporosity.

FIG. 7H is a perspective view of six films combined to make tissuescaffolds with offset cell opening patterns with a gradient pattern tocreate an interconnecting pore scaffold with a gradient in porosity inpartial section.

FIG. 8 is a flow chart depicting a method of producing a tissuescaffold.

DETAILED DESCRIPTION

We have generated tissue scaffolds that, in some embodiments, arebiocompatible (bioresorbable or nonabsorbable) scaffolds of layeredfilms, at least some of which are porous (macroporous or microporous)and that can provide controlled morphological and material-basedgradients. The films used in the scaffolds can have a structure thatprovides organization at the microstructure level that facilitatescellular invasion, proliferation, and differentiation that canultimately result in regeneration of wholly or partially functionaltissue. The films of the tissue scaffold have a gradient in compositionand microstructure that permits tissue ingrowth, tissue repair, tissueregeneration, and cell based research for therapeutic agent discovery.In particular the scaffold provides layered films that have beenmachined with openings that interface with living cells to controlgrowth in a predictable manner.

The features of such scaffolds can be controlled to suit a desiredapplication by choosing the appropriate conditions to form a layeredfilm structure with openings in select areas of each film. Thesescaffolds have distinct advantages over the prior art where thescaffolds are isotropic or random structures.

The tissue scaffolds described herein can include cell openings (e.g.,cell openings defining pores in one or more films) that vary in size andshape. Whether of a regular or irregular shape, the diameter of the cellopening can be between about 1 to about 10,000 microns. For example,cell openings can be from about 5 microns to 9,5000 microns; from about10 to 10,000 microns; from about 25 to about 7,500 microns; from about50 to 5,000 microns; from about 100 to about 2,500 microns; from about100 to about 5,000 microns; from about 250 to about 2,500 microns; fromabout 250 to about 1,000 microns; from about 500 to about 1,000 microns;from about 750 to about 1,000 microns; or ranges therebetween. Thecellular openings can provide pathways for cellular ingrowth andnutrient diffusion. Porosities can be controlled and can range fromabout 10% to 95% porous. Because the cell openings and/or channels canhave diameters in the range of microns, useful films and scaffolds canbe described as microporous. They can also be non-porous.

The features of the tissue scaffolds can be controlled to suit desiredapplications by selecting features to obtain the following properties:gradient along three axes for preferential cell culturing; channels thatrun through the scaffold for enhanced cell invasion, vascularization,and nutrient diffusion; micro-patterning of films on the surface forimproved cellular organization; tailorability of pore size and shape;anisotropic mechanical properties; composite layered structure with apolymer composition gradient to modify the cellular response todifferent materials; blends of different polymer compositions to createstructures that have portions that will degrade or resorb at differentrates; films blended or coated with bioactive agents (or “compounds”)included but not limited to biological factors, growth factors, and thelike; ability to make three dimensional structures with controlledmicrostructures; and assembly with other medical devices or agents toprovide a composite structure.

In some embodiments, a biocompatible scaffold includes a substantiallycontrollable pore structure. Characteristics selected from the groupcomprising composition, stiffness, pore architecture, and bioabsorptionrate can be controlled. The scaffold can be made from an absorbable ornonabsorbable polymers. A blend of polymers can be applied to form acompositional gradient from one layer to the next. In applications whereone composition is sufficient, the scaffold provides a biocompatiblescaffold that may have structural variations across one or more layersthat may mimic the anatomical features of the tissue. The structuralvariations can result in a variation in degradation across the scaffold.

In some embodiments, the biocompatible scaffold include interconnectingpores and channels to facilitate the transport of nutrients and/orinvasion of cells into the scaffold. Some channels may be created tofacilitate delivery of agents, compounds or cells into the scaffoldusing delivery means. Positive or negative pressure methods can beemployed to delivery the agents, compounds, or cells.

In one aspect, a method for the repair or regeneration of tissueincludes contacting a first tissue with a scaffold pore gradient at alocation on the scaffold that has appropriate characteristics to permitgrowth of the tissue. The concept of controlled transition in physicaland chemical properties, and/or microstructural features in the scaffoldcan facilitate the growth or regeneration of tissue.

The scaffolds are particularly useful for the generation of tissuejunctions between two or more layers of tissue. For a multi-cellularsystem, one type of cell can be present in one area of the scaffold anda second type of cell can be present in a separate area of the scaffold.Delivery channels can be utilized to position agents, compounds or cellsin certain regions of the scaffold. Channels can also be used togenerate controlled flow of a medium using positive or negative pressuremeans. An external source can be used to generate flow through thechannels.

A gradient of absorbable polymers of different layers forming acompositional gradient from one polymeric material to a second polymericmaterial can be created. In situations where one composition issufficient for the application, the scaffold provides a biocompatiblefilm scaffold that may have microstructural variations in the structureacross one or more dimensions that may mimic the anatomical features ofthe tissue. The cross sectional area of the implant can vary in thisinstance. When the scaffold degrades by surface erosion or through bulkdegradation, the regions with an increased cross sectional area woulddegrade at a slower rate.

The films can be layered and bonded together. The films can be attachedusing ionic or covalent bonds. Photo-initiated bonds can be createdusing suitable materials such as benzaphenone. Biocompatible adhesivescan be used. Alternatively, heat and pressure can be used.

The tissue scaffolds may be comprised of closed cell and open cellcombinations. In this instance either the closed cell or open cellfeatures may contain therapeutic agents or compounds. In addition, thedevice may comprise a stimulator that enhances the regeneration oftissue.

The materials used to produce the tissue scaffolds may be suitable forpromoting the growth of either adhering, non-adhering cell lines, or anycombination thereof.

In one case the material used to produce the scaffold comprises a sheet.The sheet may be substantially planar. The material may be at leastpartially of a layered construction. In one case the material comprisesa first layer and a second layer, the first layer having a higherabsorption rate than the second layer. The first layer may be locatedadjacent to the second layer. The second layer may be configured to belocated closer to a tissue structure than the first layer.

In one embodiment the material is at least partially porous to promotetissue in-growth. The first layer may have a higher pore density thanthe second layer. The first layer may have a smaller pore size than thesecond layer. In one case at least some of the pores form at least apartial gradient with varying density.

In another embodiment the material is at least partially porous topromote tissue in-growth. The layers may have a higher pore density inselect regions. The central region may have a higher pore density thanthe outer region. In one case at least some of the pores form at least apartial gradient from one region to the next.

The material may comprise an anti-adhesion filler filling at least someof the pores. The material may comprise an anti-adhesion coating alongat least part of the surface of the material. Alternatively, a materialused to promote tissue attachment and bonding may be utilised with thescaffold.

The scaffold can be produced by processing a biocompatible polymer intoa film and creating a controlled pore geometry in the film (i.e., thecell openings). In alternative embodiments, the film can be stretched,oriented, or otherwise manipulated (e.g., trimmed, shaped, washed orotherwise treated) before or after forming pores in the film. Where thescaffold contains more than one film, methods can be carried out byextruding a first biocompatible polymer to form a first film, extrudinga second biocompatible polymer to form a second film, attaching thefirst film to the second film to produce a implant, and forming pores inthe implant. Alternatively, the pores can be formed before the two filmsare adhered to one another. In that instance, the method of making theimplant can be carried out by: extruding a first biocompatible polymerto form a first film; forming pores in the first film; extruding asecond biocompatible polymer to form a second film; forming pores in thesecond film; and attaching the first film to the second film to producea tissue scaffold. The process can be repeated or amplified as need toproduce a scaffold having the desired number of films.

As noted, the pores can have different dimensions, the films can havedifferent thicknesses, and the films can have different compositions allof which vary the healing and biodegradation characteristics. In thatinstance, the method of making the scaffold can be carried out by:extruding a first biocompatible polymer to form a first film; formingpores in the first film; extruding a second biocompatible polymer toform a second film; forming pores in the second film; and attaching thefirst film to the second film to produce a tissue scaffold. The tissuescaffold can be designed with controlled tissue ingrowth and remodellingto permanently alter the mechanical properties of the tissue.

Where a film is obtained, rather than made, the methods of making thetissue scaffold can simply require providing a given film that is thenattached (e.g., reversibly or irreversibly bound by mechanical orchemical forces), if desired, to another film and/or processed toinclude one or more pores of a given size and arrangement. The singleprovided film (or adherent multiple films) can then be subjected to aprocess (e.g., laser ablation, die punching, or the like) that formspores within the film(s). Accordingly, any of the methods can be carriedout by providing a given biocompatible film, rather than by producing itby an extrusion or extrusion-like process. The films used in thescaffold layers can also be produced using casting, injection moulding,electrospinning, or dip coating techniques.

Preferably, the tissue scaffolds can include a film that has idealmechanical properties and a controlled thickness and that isbiocompatible. A biocompatible film is one that can, for example, residenext to biological tissue without harming the tissue to any appreciableextent. As noted above, the film(s) used in the scaffolds can includepores (e.g., open passages from one surface of the film to another) thatpermit tissue ingrowth and/or cellular infiltration.

The scaffolds can offer a combination of controlled porosity, highstrength, and specific material content, and they may have one or moreof the following advantages. They can include pores or porous structuresthat stimulate tissue integration and reduce inflammation; they canreduce the risk of rejection with adjacent tissue (this is especiallytrue with scaffolds having a smooth surface and atraumatic (e.g.,smooth, tapered, or rounded edges); they can simulate the physicalproperties of the tissue being repaired or replaced, which is expectedto promote more complete healing and minimise patient discomfort; theirsurface areas can be reduced relative to prior art devices (having areduced amount of material may decrease the likelihood of an immune orinflammatory response). Moreover, scaffolds with a reduced profile canbe produced and implanted in a minimally invasive fashion; as they arepliable, they can be placed or implanted through smaller surgicalincisions. Methods may also produce scaffolds with improved opticalproperties (e.g., scaffolds through which the surgeon can visualise moreof the underlying tissue). Practically, the micromachining techniquesthat can be used to produce the scaffolds are efficient andreproducible. The scaffolds described herein should provide enhancedbiocompatibility in a low profile configuration while maintaining therequisite strength for the intended purpose.

In one embodiment, the film is made of, or includes, a biocompatiblematerial that is biodegradable (i.e., it degrades within a human patientwithin a discernable period of time (e.g., within months or years)). Thebiocompatible material may be at least partially absorbable by the body.The biocompatible material may comprise an absorbable polymer orcopolymer such as polyglycolic acid (PGA), polylactic acid (PLA),polycaprolactone, polyhydroxyalkanoate, or polyfumarate and derivativesof the above polymers.

In another embodiment, the biocompatible material is nonabsorbable andcan be, or can include, polypropylene, polyethylene terephthalate,polytetrafluoroethylene, polyaryletherketone, nylon, fluorinatedethylene propylene, polybutester, or silicone. The tissue scaffolds canalso include a biological material such as collagen, fibrin, or elastin.Biological materials such as these can be incorporated into one or moreof the films assembled into the scaffold (e.g., as a component of thefilm or a coating thereon) or can be contained within one or more of thepores, pathways, or channels within the scaffold.

Biocompatible materials useful in the film layers can includenon-absorbable polymers such as polypropylene, polyethylene,polyethylene terephthalate, polytetrafluoroethylene,polyaryletherketone, nylon, fluorinated ethylene propylene,polybutester, and silicone, or copolymers thereof (e.g., a copolymer ofpolypropylene and polyethylene); absorbable polymers such aspolyglycolic acid (PGA), polylactic acid (PLA), polycaprolactone, andpolyhydroxyalkanoate, or copolymers thereof (e.g., a copolymer of PGAand PLA); or tissue based materials (e.g., collagen or other biologicalmaterial or tissue obtained from the patient who is to receive thescaffold or obtained from another person.) The polymers can be of theD-isoform, the L-isoform, or a mixture of both. An example of abiocompatible film suitable for producing the laminated film structureis expanded polytetrafluoroethylene.

In the case of a tissue scaffold made from film layers, the variouslayers may be of the same or different materials. For example, in thecase of an absorbable material, the material of the layers may beselected to have varying rates of absorption.

The tissue scaffolds can also include one or more materials that preventadhesions, such as hyaluronic acid. The adhesion prevention material cancoat a surface of a film, reside within one or more of the pores,pathways, or channels, or both. The adhesion prevention material maydegrade as surrounding tissue heals and minimize the risk of futureadhesions.

In one embodiment the biocompatible material has a plurality of cells.The biocompatible material may have a plurality of cells and one or moreof the cells in the plurality of cells have a diameter, measured alongthe longest axis of the cell, of about 10 to about 10,000 microns. Thebiocompatible material may have a plurality of cells and one or more ofthe cells of the plurality are essentially square, rectangular, round,oval, sinusoidal, or diamond-shaped.

In one embodiment the thickness of one or more of the films within thescaffold is about or less than about 0.25 inches. For example, thescaffold can be formed from two or more films, which can be of the sameor different thicknesses. For example, the films can be about or lessthan about 0.20 inches; about or less than about 0.18 inches; about orless than about 0.16 inches; about or less than about 0.14 inches; aboutor less than about 0.12 inches; about or less than about 0.10 inches;about or less than about 0.05 inches; about or less than about 0.025inches; about or less than about 0.020 inches; about or less than about0.015 inches; about or less than about 0.014 inches; about or less thanabout 0.013 inches; about or less than about 0.012 inches; about or lessthan about 0.011 inches; about or less than about 0.010 inches; about orless than about 0.009 inches; about or less than about 0.008 inches;about or less than about 0.007 inches; about or less than about 0.006inches; about or less than about 0.005 inches; about or less than about0.004 inches; about or less than about 0.003 inches; about or less thanabout 0.002 inches; or about 0.001 inch. In some instances, for example,where a film is non-porous, it may be thicker (e.g., about 0.5-1.0 inchthick). As noted, a given scaffold can include more than one film andthe overall thickness of the scaffold can vary tremendously, dependingon its intended application. For example, where the scaffold isimplanted to fill a void in bone or to repair a biopsy, it can be morethan an inch thick.

The tissue scaffold may comprise attachment regions, which may beadapted to receive sutures, staples or the like. In addition, theindividual layers for the tissue scaffold may have alignment regions toensure the pores in the films match up properly.

In another aspect, a method for producing a tissue scaffold, the methodcomprising: extruding a first biocompatible polymer to form a firstfilm; forming cell patterns in the first film; extruding a secondbiocompatible polymer to form a second film; forming cell patterns inthe second film; attaching the first film to the second film to producea tissue scaffold; wherein the method may further comprise the optionalstep of cleaning the scaffold.

In the case of a layered scaffold, the various layers may be of the sameor different materials. For example, in the case of an absorbablematerial, the material of the layers may be selected to have varyingrates of absorption.

Medical applications for the tissue scaffolds described above mayinclude but are not limited to tissue repair of bone, spine disc,articular cartilage, meniscus, fibrocartilage, tendons, ligaments, dura,skin, vascular grafts, nerves, liver, and pancreas. The tissue scaffoldmay be produced in a variety of shapes and sizes for the particularindication. One may select a non-absorbable scaffold for tissue defectsthat require permanent treatment and long-term durability and strength.Alternatively, one may select an absorbable scaffold for tissue defectsthat require temporary treatment when one wants to avoid the potentialcomplications associated with a permanent implant.

In addition, the tissue scaffolds can be produced in three-dimensionalforms to facilitate sizing. An example is a scaffold with a curvature toconstruct a substantially cylindrical shape. A three dimensionalstructure could be machined using a system incorporating a third axisfor micromachining. Alternatively, a substantially two-dimensionaltissue scaffold could be thermoformed into a three-dimensional shapeafter machining.

Referring collectively to FIGS. 1A-1O, film layers including cellopenings of predetermined size and configuration can be combined to forma tissue scaffold. FIG. 1A illustrates a film layer 10 used to make atissue scaffold with diamond shaped 500 micron cell openings 14 in apredetermined arrangement. The film layer 10 has known or discernabledimensions (width, length, and thickness), which can be modified or leftintact during the manufacture of a tissue scaffold. Film layer 10 is asingle layer. FIG. 1B is a side view of a film layer 10 used to make atissue scaffold. FIG. 1C is a perspective view of a film layer 18 usedto make a tissue scaffold with diamond shaped 750 micron cell openings22 in a predetermined arrangement. The film layer 18 has known ordiscernable dimensions (width, length, and thickness), which can bemodified or left intact during the manufacture of a tissue scaffold.Film layer 18 is a single layer. FIG. 1D is a side view of a film layer18 used to make a tissue scaffold. FIG. 1E is a perspective view of filmlayer 10 and film layer 18 used to make a tissue scaffold with differentoffset cell opening patterns. As shown in FIG. 1F, film layer 10 andfilm layer 18 can be combined to form a two film layer tissue scaffold26. The cell openings from the first film layer 10 are offset andinterconnect with the cell openings from the second film layer 18 tocreate a 250 micron interconnecting pore 30. FIG. 1G is a side view offilm layer 10 and film layer 18 forming a two layer tissue scaffold 26.FIGS. 1H-J are perspective views of six film layers used to make a sixlayer tissue scaffold 34. FIGS. 1K-O are perspective views of ten filmlayers used to make a ten layer tissue scaffold 38. FIG. 1O illustratesthe combination of film layers 10 and film layers 18 to create aninterconnecting pore 30.

Referring now collectively to FIGS. 2A-2H, film layers of otherembodiments including cell openings of predetermined size andconfiguration according to other embodiments can be combined to form atissue scaffold. FIG. 2A illustrates a film layer 42 used to make atissue scaffold with round 1000 micron cell openings 46 in apredetermined arrangement. The film layer 42 has known or discernabledimensions (width, length, and thickness), which can be modified or leftintact during the manufacture of a tissue scaffold. Film layer 42 is asingle layer. FIG. 2B is a side view of a film layer 42 used to make atissue scaffold. FIG. 2C is a perspective view of a film layer 50 usedto make a tissue scaffold with round 1000 micron cell openings 46 in apredetermined arrangement. It should be noted that the round cellopenings 46 have the same dimensions for film layer 42 and film layer50. The film layer 50 has known or discernable dimensions (width,length, and thickness), which can be modified or left intact during themanufacture of a tissue scaffold. Film layer 50 is a single layer. FIG.2D is a side view of a film layer 50 used to make a tissue scaffold.FIG. 2E is a perspective view of film layer 42 and film layer 50 used tomake a two layer tissue scaffold with different offset cell openingpatterns. FIG. 2F is a perspective view of film layer 42 and film layer50 used to make a six layer tissue scaffold with different offset cellopening patterns. FIG. 2G is a perspective view of film layers 42 andfilm layers 50 forming a six layer tissue scaffold 54. FIG. 2Hillustrates the combination of film layers 42 and film layers 50 tocreate an interconnecting pore 58. The round 1000 micron cell openings46 from the first film layer 42 are offset by 750 microns andinterconnect with the round 1000 micron cell openings 46 from the secondfilm layer 50 to create a 250 micron interconnecting pore 58.

Referring collectively to FIGS. 3A-3M, film layers of furtherembodiments including cell openings of predetermined size andconfiguration according to other embodiments can be combined to form atissue scaffold. FIGS. 3A-J illustrate film layers 62, 66, 70, 74, and78 used to make a tissue scaffold with round shaped cell openings 82 andoval shaped cell openings 86 in a predetermined arrangement. The roundshaped cell openings 82 and oval shaped cell openings 86 have diametersranging from 300 microns to 1300 microns. The film layers 62, 66, 70,74, and 78 have known or discernable dimensions (width, length, andthickness), which can be modified or left intact during the manufactureof a tissue scaffold. The film layers have alignment holes 90 to ensurethe cell openings from each film layer are offset in a controlledmanner. FIG. 3K is a perspective view of five film layers 62, 66, 70,74, and 78 used to make a tissue scaffold with offset cell openingpatterns. FIG. 3L is perspective view of the five film layers 62, 66,70, 74, and 78 repeated five times to make a twenty five layer tissuescaffold 94. FIG. 3M illustrates the combination of offset film layers62, 66, 70, 74, and 78 with cell openings to create a network ofinterconnecting pores 98. The outer section containing the alignmentholes 90 has been removed from the tissue scaffold.

Referring collectively to FIGS. 4A-4I, film layers of some embodimentsincluding cell openings of predetermined size and configurationaccording to other embodiments can be combined to form a tissuescaffold. FIG. 4A illustrates a film layer 102 used to make a tissuescaffold with oval cell openings 106 with diameters ranging from 60 to950 microns in a predetermined arrangement. The predeterminedarrangement has a gradient of higher porosity on the edge to lowerporosity in the center. The film layer 102 has known or discernabledimensions (width, length, and thickness), which can be modified or leftintact during the manufacture of a tissue scaffold. Film layer 102 is asingle layer. FIG. 4B is a side view of a film layer 102 used to make atissue scaffold. FIG. 4C illustrates a film layer 110 used to make atissue scaffold with oval cell openings 114 with diameters ranging from270 to 2080 microns in a predetermined arrangement. The predeterminedarrangement has a gradient of higher porosity on the edge to lowerporosity in the center. The film layer 110 has known or discernabledimensions (width, length, and thickness), which can be modified or leftintact during the manufacture of a tissue scaffold. Film layer 110 is asingle layer. FIG. 4D is a side view of a film layer 110 used to make atissue scaffold. FIG. 4E is a perspective view of film layer 102 andfilm layer 110 used to make a two layer tissue scaffold with differentoffset cell opening patterns. FIG. 4F is a perspective view of filmlayer 102 and film layer 110 used to make a two layer tissue scaffold118 with different offset cell opening patterns. FIG. 4G is a side viewof film layers 102 and film layers 110 forming a two layer tissuescaffold 118. The predetermined arrangement of the cell openings createsa gradient of higher porosity on the edge to lower porosity in thecenter which, in turn, creates a gradient of interconnecting pores withhigher interconnecting areas on the edge of the tissue scaffold 118.FIGS. 4H-I are perspective views of ten film layers used to make a tenlayer tissue scaffold 122.

Referring collectively to FIGS. 5A-5C, film layers of furtherembodiments including cell openings and hydrogel film layers can becombined to form a tissue scaffold. FIG. 5A illustrates fifteen filmlayers 102 and 110 used to make a tissue scaffold with offset cellopening patterns with a gradient pattern and two hydrogel film layers126. Film layer 102 is used with film layer 110 to make a tissuescaffold assembly. Hydrogel film layer 126 is place under film layer 102during assembly of the tissue scaffold. The hydrogel film layer canpromote cellular interaction with the tissue scaffold with or withoutthe use of therapeutic agents. FIGS. 5B-C are perspective views offifteen film layers used to make a tissue scaffold 130 with offset cellopening patterns forming interconnecting pores 134 with a gradientpattern and two hydrogel film layers 126. The outer section containingthe alignment holes 90 has been removed from the tissue scaffold.

Referring collectively to FIGS. 6A-6K, film layers including deliverychannel openings are combined to form a tissue scaffold. FIG. 6Aillustrates twenty seven film layers 138 used to make a tissue scaffoldwith delivery channels 146. FIG. 6B is a perspective view of twenty fivefilm layers 138 used to make a tissue scaffold with delivery channels.FIG. 6C is a side view of a film layers 138 used to make a tissuescaffold. FIG. 6D is a perspective view of twenty five film layers 138combined to make a tissue scaffold with delivery channels within thescaffold with a cut out section depicting large delivery channels 150and small delivery channels 154. FIG. 6E is a perspective view of twentyseven film layers combined to make a tissue scaffold 146 with deliverychannel openings within the tissue scaffold. Alignment holes 90 anddelivery channel openings 158 for delivery agents or cells are alsoshown with a cut out section. FIG. 6F is a perspective view of twentyseven films combined to make a tissue scaffold with delivery channelopenings 158 containing an injector within the scaffold with a cut outsection depicting the process of delivering agents or cells 162 to thechannel openings. FIG. 6G is a perspective view of twenty seven filmscombined to make a tissue scaffold with delivery channel openings withinthe scaffold with a cut out section depicting delivered agents or cells162 to the channel openings. FIG. 6H is a perspective view of twoseparate tissue scaffolds 166 and 170 with separate delivery channelopenings within each scaffold with a cut out section depicting deliveredagents or cells to the channel openings. FIG. 6I is a perspective viewof two separate tissue scaffolds 166 and 170 combined to make acomposite tissue scaffold 174 with separate delivery channel openingswithin each scaffold with a cut out section depicting delivered agentsor cells to the channel openings. FIG. 6J is a perspective sectionedview of the twenty five film layer tissue scaffold combined to make atissue scaffold with ingrowth channel openings 178 within the scaffoldthat extend to the center of the scaffold. FIG. 6K is a perspectivesectioned view of twenty five film layer tissue scaffold combined tomake a tissue scaffold with ingrowth channel openings 178 within thescaffold that extend to the center of the scaffold. The tissue scaffoldis depicted with a blood vessel 182 growing into the ingrowth channelopenings 178 of the scaffold.

Referring collectively to FIGS. 7A-7H, film layers of still furtherembodiments including cell openings of predetermined size andconfiguration are combined to form a tissue scaffold. FIG. 7Aillustrates a film layer 186 used to make a tissue scaffold with ovalcell openings 190 in a predetermined arrangement. The predeterminedarrangement has a gradient of higher porosity in select regions to lowerporosity in select regions. The film layer 186 has known or discernabledimensions (width, length, and thickness), which can be modified or leftintact during the manufacture of a tissue scaffold. Film layer 186 is asingle layer. FIG. 7B is a side view of a film layer 186 used to make atissue scaffold. FIG. 7C illustrates a film layer 194 used to make atissue scaffold with oval cell openings 190 in a predeterminedarrangement. The predetermined arrangement has a gradient of higherporosity in select regions to lower porosity in select regions. The filmlayer 194 has known or discernable dimensions (width, length, andthickness), which can be modified or left intact during the manufactureof a tissue scaffold. Film layer 194 is a single layer. FIG. 7D is aside view of a film layer 194 used to make a tissue scaffold. FIG. 7E isa perspective view of film layer 186 and film layer 194 used to make atwo layer tissue scaffold with different offset cell opening patterns.FIG. 7F is a perspective view of film layer 186 and film layer 194 usedto make a six layer tissue scaffold with different offset cell openingpatterns. FIGS. 7G-H are perspective views of film layers 186 and filmlayers 194 forming a six layer tissue scaffold 198. The predeterminedarrangement of the cell openings creates a gradient of higher porosityin select regions to lower porosity in select regions which, in turn,creates a gradient of interconnecting pores 202 with higherinterconnecting areas in select regions of the tissue scaffold 198.

FIG. 8 is a flow chart illustrating some of the steps in a method ofproducing a tissue scaffold of the present invention.

EXAMPLES Example 1

A tissue scaffold was constructed using a copolymer film of polylacticacid (PLA) and polycaprolactone (PCL). A tissue scaffold measuring 6000microns in thickness was fabricated by combining six 1000 micron thickfilm layers with round 1000 micron cell openings. Three of the films hadthe cell pattern depicted in FIG. 2A and three of the films had the cellpattern depicted in FIG. 2C. A 100 watt CO² laser was used to create thecell openings in the film layers using a CAD-CAM process. Because thecell openings in film layers were machined to the specificationsoutlined in FIGS. 2A and 2C, the cell openings were offset by 750microns. Consequently, interconnecting pores measuring 250 microns werecreated between the cell openings in the film layers. Six film layerswere built up to create the scaffold. A 4061 cyanoacrylate adhesivemanufactured by Henkel Loctite (Hertfordshire, UK) was used to bond thefilm layers together.

Example 2

The scaffold created in Example 1 can be used as a scaffold forevaluating cellular behaviour in a three-dimensional environment. Thescaffold can be included in a kit that includes a sterile polystyrenetissue culture plate with the standard number of wells 6, 12, 24, 48 or96 within which the scaffolds have been placed, instructions for thecellular seeding and/or optimal dispersion concentration ofgrowth/active factors, and accessory tools for proper scaffold handling.In a different approach, the invention can feature a kit that includessterile pre-formed three-dimensional scaffold shapes, a lyophilized or acombination of lyophilized growth/active factor(s), associated tools toallow the delivery of the lyophilized agents homogenously within thescaffold, and instructions for proper growth/active factor dispersion.In a different approach, the invention can feature a kit that includessterile pre-formed 3D scaffold shapes, a lyophilized or a combination oflyophilized growth/active factor(s), a photopolymerizable agent, a vialto mix the photopolymerizable agent with the lyophilized compound,associated tools to allow the homogenous distribution of thephotopolymerizable agent plus lyophilized compound into the scaffold,and necessary instructions. The kit could or could not include a lightsource to induce local photopolymerization, thus, trapping of thelyophilized compound into the 3D scaffold.

A number of embodiments have been described. Other embodiments arewithin the scope of the following claims.

What is claimed is:
 1. A tissue scaffold comprising: a first filmcomprising a plurality of cell openings; and a second film adjacent thefirst film and comprising a plurality of cell openings offset in apredetermined arrangement from the cell openings of the first film;wherein the cell openings of the first film interconnect with the cellopenings of the second film to define pathways with interconnectingpores of controlled area extending from the first film to the secondfilm; and wherein the diameter of a majority of the pores is less thanthe diameter of the cell openings of the first and second films thatdefine each pore.
 2. The tissue scaffold of claim 1, further comprisinga plurality of delivery channels extending from the first film to thesecond film.
 3. The tissue scaffold of claim 1, wherein at least one ofthe first and second films further comprises alignment holes to alignthe cell openings of the first and second films when joined together. 4.The tissue scaffold of claim 1, wherein at least one of the cellopenings comprises a therapeutic agent and, optionally, at least one ofthe films is an oriented film.
 5. The tissue scaffold of claim 1,wherein the first and second films are attached together using abiocompatible adhesive.
 6. The tissue scaffold of claim 1, wherein thefirst and second films are substantially circular.
 7. The tissuescaffold of claim 6, wherein at least one of the first and second filmscomprises progressively larger cell openings along a radial direction todefine a cell opening gradient.
 8. The tissue scaffold of claim 1,wherein at least one of the first and second films comprises a pluralityof cell openings sized and configured to define a cell opening gradientalong the film.
 9. The tissue scaffold of claim 1, wherein the firstfilm comprises a first material and the second film comprises a secondmaterial, wherein the first material has a higher absorption rate thanthe second material.
 10. The tissue scaffold of claim 1, wherein in afirst orientation of the first film with respect to the second film, thecell openings of the first film are aligned with the cell openings ofthe second film to define a first plurality of pathways and wherein in asecond orientation of the first film with respect to the second film,the cell openings of the first film are substantially offset from thecell openings of the second film to define a second plurality ofpathways.
 11. The tissue scaffold of claim 1, wherein the cell openingsof the first and second films are sized and configured to definemultiple cell opening gradients to establish pathways for preferentialcell culturing.
 12. The tissue scaffold of claim 1, wherein the cellopenings include a diameter between about 10 to about 10,000 microns.13. The tissue scaffold of claim 1, wherein at least one of the firstand second films comprises attachment regions configured to receivesurgical fastening elements.
 14. The tissue scaffold of claim 1, whereinat least one of the cell openings comprises a material to substantiallysuppress tissue adhesion and at least one of the cell openings comprisesa material to substantially promote tissue adhesion.
 15. The tissuescaffold of claim 1, wherein the cell openings of the second film areoffset from the cell openings of the first film to form a combination ofclosed cells and open cells.
 16. The tissue scaffold of claim 1, whereinthe scaffold is greater than 750 microns thick.
 17. The tissue scaffoldof claim 1, wherein the scaffold is greater than 0.5 inches thick.
 18. Atissue scaffold comprising: a first film including a first porosity; anda second film joined to the first film and including a second porositygreater than the first porosity; and a plurality of delivery channelsextending through the first and second films in a predeterminedarrangement; wherein the first porosity and the second porosity define acontrolled porosity gradient extending from the first to the second filmto selectively promote cellular regeneration along the gradient; andwherein the diameter of a majority of the delivery channels is less thanthe diameter of the cell openings of the first and second films thatdefine each channel.
 19. A tissue scaffold comprising: a first filmincluding a plurality of cell openings; and a second film adjacent thefirst film, the second film including a plurality of cell openings andalignment holes; wherein the alignment holes align the cell openings ofthe first film with the cell openings of the second film such that thecell openings of the first film interconnect with the cell openings ofthe second film to define pathways with interconnecting pores ofcontrolled area extending from the first film to the second film; andwherein the diameter of a majority of the pores is less than thediameter of the cell openings of the first and second films that defineeach pore.
 20. The tissue scaffold of claim 19, wherein at least one ofthe first and second films comprises a plurality of cell openings sizedand configured to define a cell opening gradient along the film.
 21. Thetissue scaffold of claim 19, wherein in a first orientation of the firstfilm with respect to the second film, the cell openings of the firstfilm are aligned with the cell openings of the second film to define afirst plurality of pathways and wherein a second orientation of thefirst film with respect to the second film, the cell openings of thefirst film are substantially offset from the cell openings of the secondfilm to define a second plurality of pathways with interconnecting poresof controlled area extending from the first film to the second film. 22.The tissue scaffold of claim 19, wherein at least one of the first andsecond films comprises attachment regions configured to receive surgicalfastening elements.
 23. The tissue scaffold of claim 19, wherein thecell openings of the second film are offset from the cell openings ofthe first film to form a combination of closed cells and open cells. 24.The tissue scaffold of claim 19, wherein the scaffold is greater than750 microns thick.
 25. The tissue scaffold of claim 19, wherein thescaffold is greater than 0.5 inches thick.